Linking the structure and thermal stability of beta-galactoside-binding protein galectin-1 to ligand binding and dimerization equilibria.

The stability of proteins involves a critical balance of interactions of different orders of magnitude. In this work, we present experimental evidence of an increased thermal stability of galectin-1, a multifunctional beta-galactoside-binding protein, upon binding to the disaccharide lactose. Analysis of structural changes occurring upon binding of lectin to its specific glycans and thermal denaturation of the protein and the complex were analyzed by circular dichroism. On the other hand, we studied dimerization as another factor that may induce structural and thermal stability changes. The results were then complemented with molecular dynamics simulations followed by a detailed computation of thermodynamic properties, including the internal energy, solvation free energy, and conformational entropy. In addition, an energetic profile of the binding and dimerization processes is also presented. Whereas binding and cross-linking of lactose do not alter galectin-1 structure, this interaction leads to substantial changes in the flexibility and internal energy of the protein which confers increased thermal stability to this endogenous lectin. Given that an improved understanding of the physicochemical properties of galectin-glycan lattices may contribute to the dissection of their biological functions and prediction of their therapeutic applications, our study suggests that galectin binding to specific disaccharide ligands may increase the thermal stability of this glycan-binding protein, an effect that could influence its critical biological functions.

Critical role of the solvent environment in galectin-1 binding to the disaccharide lactose.

Galectin-1 (Gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, binds specifically to poly-N-acetyllactosamine-enriched glycoconjugates. Through interactions with these glycoconjugates, this protein modulates inflammatory responses and contributes to tumor progression and immune cell homeostasis. The carbohydrate recognition domain includes the single protein tryptophan (Trp68). UV resonance Raman spectroscopy and molecular dynamic simulation were used to examine the change in the environment of the Trp on ligand binding. The UV Raman spectra and the calculated water radial distribution functions show that, while no large structural changes in the protein follow lactose binding, substantial solvent reorganization occurs. These new insights into the microscopic role of water molecules in Gal-1 binding to its specific carbohydrate ligands provides a better understanding of the physicochemical properties of Gal-1-saccharide interactions, which will be useful for the design of synthetic inhibitors for therapeutic purposes.

Characterization of the galectin-1 carbohydrate recognition domain in terms of solvent occupancy.

Human galectin-1, a galactosil-terminal sugar binding soluble protein, is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein interactions. Recent studies revealed that it plays a key role as a modulator of cellular differentiation and immunological response. In this work, we have investigated the solvation properties of the carbohydrate recognition domain of Gal-1 by means of molecular dynamics simulations. Water sites (ws) were identified in terms of radial and angular distribution functions, and properties such as water residence times, interaction energies, and free-energy contributions were evaluated for those sites. Our results allowed us to correlate the thermodynamic properties of the ws and their binding pattern with the N-acetilgalactoside ligand. These results let us further infer that the water molecules located at the ws, which exhibit much more favorable binding, are the ones replaced by -OH groups of the sugar.

Identification of an equilibrium intermediate in the unfolding process of galectin-1, which retains its carbohydrate-binding specificity.

The unfolding process of galectin-1 (Gal-1) in the presence of a denaturing agent was examined using fluorescence and far-UV circular dichroism (CD) spectroscopy determinations, and was found to be completely reversible. The data showed that the transitions of guanidine hydrochloride (GdnHCl)-induced lectin unfolding, in the absence of ligand, were biphasic in nature, clearly showing the existence of at least one stable intermediate. On the other hand, the unfolding in the presence of disaccharide yielded data that could fit very well to a two-state model, indicating a stabilizing effect of the ligand. The folding intermediate was further characterized by size exclusion chromatography, near-UV CD and anilinonaphtalene sulfonate binding, and shown to belong to the molten globule type. Strikingly, this intermediate retained its carbohydrate-binding specificity, as evidenced by the tryptophan fluorescence changes detected upon its interaction with lactose.